Differential Codon Usage Pattern of HIV-1 tat Gene in Those with Slower and Faster Rates of Disease Progression.

Codon usage has been identified as one of the most important factors that influence gene expression. The frequencies with which the different codons are used vary significantly between different organisms and also between the genes within the same organism. HIV has a remarkable nucleotide composition with an above average percentage of "A" nucleotides resulting in a codon usage pattern different from that of the human host. In this study, we compared the codon usage pattern of HIV-1 genes among different groups of HIV disease progressors to understand the influence of differential codon usage pattern on the pathogenic manifestation in the host.

Delivery of PUMA Apoptosis Gene Using Polyethyleneimine-SMCC-TAT/DNA Nanoparticles: Biophysical Characterization and In Vitro Transfection Into Malignant Melanoma Cells.

A synthesized PEI-based gene delivery system, wherein PEI was crosslinked with sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) conjugating trans-activating transcriptional activator (TAT), yielding PEI-SMCC-TAT (PST), a novel non-viral vector for apoptosis-related gene PUMA (p53 up regulated modulator of apoptosis), was designed and evaluated. Sulfo-SMCC is a commonly used heterobifunctional crosslinker and is soluble in water, making the crosslinking easier without organic reagent like DMSO or chloroform. The PST/pDNA nanoparticles were 171.9 nm at the optimal N/P ratio (50:1). DNA complexes of all the PST conjugation had much lower toxicity and exhibited enhancement in transfection efficiency in comparison with single PEI vector. The results also showed that the transfection efficiency of PST/pEGFP nanoparticles into malignant melanoma A375 cell increased, and PST carrying PUMA gene induced the apoptosis of A375 cells. It was suggested that PST could be a promising melanoma tumor-targeting nanovector, and have a good potential in clinical application.

A gene therapy approach to eliminate HIV-1-infected cells.

The ideal therapy for HIV infection requires a method to eliminate all HIV-harboring cells in the infected individual. The authors are developing an HIV-specific promoter to drive the expression of suicide genes that would induce cell death specifically in HIV-infected cells. The authors constructed a promoter that is 100-fold more responsive to the HIV transcriptional activator, Tat, than cellular transcription factors, using a plasmid expressing luciferase under the control of the mutated LTR promoter.

Genetic characterization of HIV type 1 Tat exon 1 from a southern Indian clinical cohort: identification of unique epidemiological signature residues.

The trans-activator of transcription (Tat) of HIV-1 plays an important role in viral infection and pathogenesis. We examined the genetic characteristics of exon 1 of the tat gene derived from 102 seropositive subjects from southern India. Database-derived Indian (n=105) and global (n=413) HIV-1C sequences were also used for viral epidemiological signature pattern analysis in the Tat open reading frame (ORF). We identified HIV-1C as the most predominant genetic subtype (99%) and the presence of a novel A1C recombinant strain in one study participant. After examining all the available HIV-1C Indian sequences from primary clinical isolates and database-derived sequences, we found a high level of sequence conservation (92.6 ± 12%) within Tat amino acid residues. Furthermore, signature pattern analysis identified five amino acid positions in Tat that contained signature residues unique for Indian HIV-1C consisting of 21A, 24N, 29K, 40K, and 60Q. Our data have direct relevance for subunit-based Tat HIV-1 vaccine development.

"Soft" calcium crosslinks enable highly efficient gene transfection using TAT peptide.

PURPOSE: Typically, low molecular weight cationic peptides or polymers exhibit poor transfection efficiency due to an inability to condense plasmid DNA into small nanoparticles. Here, efficient gene delivery was attained using TAT/pDNA complexes containing calcium crosslinks. METHODS: Electrostatic complexes of pDNA with TAT or PEI were studied with increasing calcium concentration. Gel electrophoresis was used to determine DNA condensation. The morphology of the complexes was probed by transmission electron microscopy. Transfection efficiency was assessed using a luciferase reporter plasmid. The accessibility of phosphate and amine groups within complexes was evaluated to determine the effect of calcium on structure. RESULTS: TAT/pDNA complexes were condensed into small, 50-100 nm particles by optimizing the concentration of calcium. Complexes optimized for small size also exhibited higher transfection efficiency than PEI polyplexes in A549 cells. TAT and TAT complexes displayed negligible cytotoxicity up to 5 mg/mL, while PEI exhibited high cytotoxicity, as expected. Probing the TAT-Ca/pDNA structure suggested that calcium interacted with both phosphate and amine groups to compact the complexes; however, these "soft" crosslinks could be competitively disrupted to facilitate DNA release. CONCLUSION: Small and stable TAT-Ca/pDNA complexes were obtained via "soft" calcium crosslinks leading to sustained gene expression levels higher than observed for control PEI gene vectors. TAT-Ca/pDNA complexes were stable, maintaining particle size and transfection efficiency even in the presence of 10% of FBS. TAT-Ca complexes offer an effective vehicle offering potential for translatable gene delivery.

HIV-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splicing and virus production in macrophages.

BACKGROUND: Macrophages are important targets and long-lived reservoirs of HIV-1, which are not cleared of infection by currently available treatments. In the primary monocyte-derived macrophage model of infection, replication is initially productive followed by a decline in virion output over ensuing weeks, coincident with a decrease in the levels of the essential viral transactivator protein Tat. We investigated two possible mechanisms in macrophages for regulation of viral replication, which appears to be primarily regulated at the level of tat mRNA: 1) differential mRNA stability, used by cells and some viruses for the rapid regulation of gene expression and 2) control of HIV-1 alternative splicing, which is essential for optimal viral replication. RESULTS: Following termination of transcription at increasing times after infection in macrophages, we found that tat mRNA did indeed decay more rapidly than rev or nef mRNA, but with similar kinetics throughout infection. In addition, tat mRNA decayed at least as rapidly in peripheral blood lymphocytes. Expression of cellular splicing factors in uninfected and infected macrophage cultures from the same donor showed an inverse pattern over time between enhancing factors (members of the SR family of RNA binding proteins) and inhibitory factors (members of the hnRNP family). While levels of the SR protein SC35 were greatly up-regulated in the first week or two after infection, hnRNPs of the A/B and H groups were down-regulated. Around the peak of virus production in each culture, SC35 expression declined to levels in uninfected cells or lower, while the hnRNPs increased to control levels or above. We also found evidence for increased cytoplasmic expression of SC35 following long-term infection. CONCLUSION: While no evidence of differential regulation of tat mRNA decay was found in macrophages following HIV-1 infection, changes in the balance of cellular splicing factors which regulate alternative viral pre-mRNA splicing were observed. These changes correlated with changes in Tat expression and virus production and could play an important role in viral persistence in macrophages. This mechanism could provide a novel target for control of infection in this critical cell type, which would be necessary for eventual eradication of the virus from infected individuals.

TAT peptide-modified liposomes provide enhanced gene delivery to intracranial human brain tumor xenografts in nude mice.

In this study, we have investigated the potential of trans-activating transcriptional activator peptide (TATp)-modified liposomes to enhance the delivery of the model gene, plasmid encoding for the green fluorescent protein (pEGFP-N1), to human brain tumor U-87 MG cells in vitro and in an intracranial model in nude mice. The TATp-lipoplexes were characterized at lipid/DNA (+/-) charge ratios of 0.2, 5, 10, and 20 for size analysis and DNA complexation. The size distribution of DNA-loaded TATp-liposomes was narrow and the DNA complexation was firm at lipid/DNA (+/-) charge ratios of 5 and higher. TATp-lipoplexes had demonstrated an enhanced delivery of pEGFP-N1 to U-87 MG tumor cells in vitro at lipid/DNA (+/-) charge ratios of 5 and 10. In vivo transfection of intracranial brain tumors by intratumoral injections of TATp-lipoplexes showed an enhanced delivery of pEGFP-N1 selectively to tumor cells and subsequent effective transfection compared to plain plasmid-loaded lipoplexes. No transfection (green fluorescence of the GFP) was noted in the normal brain adjacent to tumor.

Safety and efficacy of a lentiviral vector containing three anti-HIV genes--CCR5 ribozyme, tat-rev siRNA, and TAR decoy--in SCID-hu mouse-derived T cells.

Gene therapeutic strategies show promise in controlling human immunodeficiency virus (HIV) infection and in restoring immunological function. A number of efficacious anti-HIV gene constructs have been described so far, including small interfering RNAs (siRNAs), RNA decoys, transdominant proteins, and ribozymes, each with a different mode of action. However, as HIV is prone to generating escape mutants, the use of a single anti-HIV construct would not be adequate to afford long range-viral protection. On this basis, a combination of highly potent anti-HIV genes--namely, a short hairpin siRNA (shRNA) targeting rev and tat, a transactivation response (TAR) decoy, and a CCR5 ribozyme--have been inserted into a third-generation lentiviral vector. Our recent in vitro studies with this construct, Triple-R, established its efficacy in both T-cell lines and CD34 cell-derived macrophages. In this study, we have evaluated this combinatorial vector in vivo. Vector-transduced CD34 cells were injected into severe combined immunodeficiency (SCID)-hu mouse thy/liv grafts to determine their capacity to give rise to T cells. Our results show that phenotypically normal transgenic T cells are generated that are able to resist HIV-1 infection when challenged in vitro. These important attributes of this combinatorial vector show its promise as an excellent candidate for use in human clinical trials.

High frequency of defective vpu compared with tat and rev genes in brain from patients with HIV type 1-associated dementia.

Human immunodeficiency virus type 1 (HIV) infection of the central nervous system frequently causes HIV-associated dementia (HAD) and other neurological disorders. The role of HIV regulatory and accessory proteins in the pathogenesis of these disorders is unclear. Here we analyzed sequences of tat, rev, and vpu genes in 55 subgenomic clones previously shown to encode functional env genes from brain and lymphoid tissues of four AIDS patients with HAD. Phylogenetic analysis showed distinct compartmentalization of tat, rev, and vpu genes in brain versus lymphoid tissues. Nine of 19 vpu sequences from brain of two patients had premature stop codons at positions between amino acids 2 and 30, compared with 0 of 8 from lymphoid tissues. Tat sequences from brain (n = 8 of 8) but not lymphoid (n = 0 of 6) tissue from one patient had a 35 amino acid truncation at the C-terminus. Rev sequences from the brain of one patient (n = 6 of 8) had a 5 amino acid truncation. These results demonstrate a high frequency of defective vpu compared with tat and rev genes in brain from HAD patients, and identify sequence variants of these regulatory/accessory genes that may influence the pathogenesis of HIV-associated neurological disease.

The Effect of a Single Nucleotide Substitution in the Splicing Silencer in the tat/rev Intron on HIV Type 1 Envelope Expression.

A complex mRNA splicing pattern, which remains to be fully characterized, influences HIV-1 gene expression. In this study, poor envelope expression of a primary HIV-1 isolate was observed and linked to increased splicing of the two coding exons of tat/rev. The substitution of a nucleotide G, located 28 nucleotides upstream of the splice acceptor site SA7 in the recently identified intron splicing silencer sequence, was found to be responsible for the poor envelope expression. A single nucleotide substitution of G with A at this position results in a poor envelope expression phenotype. Moreover, substitution of the nucleotide G with any other nucleotide in an infectious HIV-1 proviral clone, HXB2RU3, results in poor envelope expression. The substitution of this nucleotide reduces the hnRNP A1 binding affinity but increases the splicing of env mRNA. The nucleotide G at this position is highly conserved among HIV-1 isolates and appears to play a critical role in HIV-1 splicing.

Phylogenetic analysis of env, gag, and tat genes of HIV type 1 detected among the injecting drug users in West Bengal, India.

A recent occurrence of HIV-1 seropositivity among a group of injecting drug users (IDUs) in Darjeeling, a hilly district in northern West Bengal, revealed overall 11.8% HIV seroprevalence. Our study based on env (C2-V3), gag (p24-p7), and tat (exon-1) genomic regions of HIV-1 detected among this population showed that Darjeeling IDU sequences belonged to subtype C. Interestingly, the IDU sequences from Darjeeling were again found to be closer to the C strains from Manipur, a northeastern state in India, which is linked to the Golden Triangle via the Manipur-Myanmar border, rather than the IDU C sequences from Nepal, a neighboring country of India. The outgroup reference strains from different sites of IDU-driven epidemics in the world like Russia, Vietnam, Thailand, and Spain belonged to the nonsubtype C group and formed separate clusters from the subtype C cluster in our analysis. These results indicate a rapid spread of HIV-1 by possible drug trafficking along international boundaries, which might also help in the invasion of HIV-1 among IDUs of Darjeeling through the Manipur-Myanmar border of India.

[Study on the relationship between the polymorphisms and secondary structure of tat exon-1 gene and HIV/ AIDS progress in subtype B' and B'/C].

OBJECTIVE: To study the polymorphisms and secondary structure of human immunodeficiency virus (HIV-1) tat exon 1 among subtype B' and B'/C HIV-1 infected people in China and to explore the relationship between the polymorphism of tat exon 1 and the disease progression. METHODS: 8 subtype B' and 5 B'/C HIV-1 infected patients with slow disease progression were selected from Liaoning, Jilin and Yunnan province. 26 subtype B' and 9 B'/C HIV-1 infected patients with similar sex, age but with typical disease progression were selected. Provirus was extracted from the whole blood. The gene sequences of the Tat exon 1 were amplified by nest-polymerase chain reaction (nest-PCR). Products were purified and sequenced directly. The sequences were aligned, translated, amino acid substitution were analyzed and secondary structures were predicted. RESULTS: Many amino acid substitution could be found in the exon 1 of Tat in HIV-1 subtype B' and B'/C recombinant strain infected persons with different disease progression except A58T,none of them showed definitely relationship with HIV viral load and disease progression. 23N, 31S, 32Y and 46F were subtype-specific substitutions. No characteristic secondary structure of exon 1 of Tat was found. CONCLUSION: Some of the mutations of tat exon 1 might be related to HIV viral load and disease progression. However, there was no relationship found between the secondary structure of Tat protein and the disease progression.

Linear double-stranded DNA that mimics an infective tail of virus genome to enhance transfection.

Our previous work showed that a natural beta-(1-->3)-d-glucan schizophyllan (SPG) can form a stable complex with single-stranded oligonucleotides (ssODNs). When protein transduction peptides were attached to SPG and this modified SPG was complexed with ssODNs, the resultant complex could induce cellular transfection of the bound ODNs, without producing serious cytotoxicity. However, no technique was available to transfect double-stranded DNAs (dsDNA) or plasmid DNA using SPG. This paper presents a new approach to transfect dsDNA, showing preparation and transfection efficiency for a minimal-size gene having a loop-shaped poly(dA)(80) on both ends. This poly(dA) loops of dsDNA can form a complex with SPG. An siRNA-coding dsDNA with the poly(dA) loop was complexed with Tat-attached SPG to silence luciferase expression. When LTR-Luc-HeLa cells that can express luciferase under the control of the LTR promoter were exposed to this complex, the expression of luciferase was suppressed (i.e., RNAi effect was enhanced). Cytotoxicity studies showed that the Tat-SPG complex induced much less cell death compared to polyethylenimine, indicating that the proposed method caused less harm than the conventional method. The Tat-SPG/poly(dA) looped dsDNA complex had a structure similar to the viral genome in that the dsDNA ends were able to induce transfection and protection. The present work identifies the SPG and poly(dA) looped minimum-sized gene combination as a candidate for a non-toxic gene delivery system.

Nuclear targeting of protein phosphatase-1 by HIV-1 Tat protein.

Transcription of human immunodeficiency virus (HIV)-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of the C-terminal domain of RNA polymerase-II by CDK9/cyclin T1. We previously showed that Tat-induced HIV-1 transcription is regulated by protein phosphatase-1 (PP1). In the present study we demonstrate that Tat interacts with PP1 and that disruption of this interaction prevents induction of HIV-1 transcription. We show that PP1 interacts with Tat in part through the binding of Val36 and Phe38 of Tat to PP1 and that Tat is involved in the nuclear and subnuclear targeting of PP1. The PP1 binding mutant Tat-V36A/F38A displayed a decreased affinity for PP1 and was a poor activator of HIV-1 transcription. Surprisingly, Tat-Q35R mutant that had a higher affinity for PP1 was also a poor activator of HIV-1 transcription, because strong PP1 binding competed out binding of Tat to CDK9/cyclin T1. Our results suggest that Tat might function as a nuclear regulator of PP1 and that interaction of Tat with PP1 is critical for activation of HIV-1 transcription by Tat.

The role of the Tat gene in the pathogenesis of HIV infection.

The human immunodeficiency virus Tat regulatory protein is essential for virus replication and for the efficient transcription of HIV-1 provirus, and in the pathogenesis of AIDS. The role of the tat gene was investigated in 300 samples. It was found that 71.7% were subtype CRF_01AE, 9.3% were subtype B, while 11.7 and 7.3% of them were cross-reactive and non-typeable, respectively. Moreover the results from peptide ELISA also showed that a low CD4 cell count was related to a low anti-Tat antibody (p < 0.05), which may be due to the progression of HIV-1, which can be found predominantly in AIDS patients. The results of nested PCR showed that the second Tat exon might also play a role in T-cell activation. Reverse transcription polymerase chain reaction (RT-PCR) was used to measure HIV-1 mRNA expression in PBMC. RT-PCR negative results were found mostly in the asymptomatic HIV-seropositive group (88%). HIV-1 mRNA expression was found to correlate with current immunologic status. The differences in Tat protein sequences from DNA sequencing between the patients who had anti-Tat antibody positive and anti-Tat antibody negative, were not significant (p > 0.05). These results suggested that the Tat amino acid sequences were conserved among each group of samples and did not change significantly compared with the consensus sequence in previous studies. Several factors make Tat an attractive target for vaccine design.

Functionally-inactive and immunogenic Tat, Rev and Nef DNA vaccines derived from sub-Saharan subtype C human immunodeficiency virus type 1 consensus sequences.

The efficacy of cellular immune responses elicited by HIV vaccines is dependent on their strength, durability and antigenic breadth. The regulatory proteins are abundantly expressed early in the viral life cycle and CTL recognition may bring about early killing of infected cells. We synthesised DNA vaccine constructs that encode consensus HIV-1 subtype C Tat, Rev and Nef proteins. Proteins carrying inactivating mutations were tested for functional activity and highly expressing, inactive Tat, Rev and Nef mutants were identified and their reading frames fused into a TatRevNef cassette. Single- and polygene Tat, Rev and/or Nef constructs were immunogenic in BALB/c mice. These constructs may serve to increase the antigenic breadth for an HIV-1 vaccine that is relevant for sub-Saharan Africa.

Determinants of human immunodeficiency virus type 1 escape from the primary CD8+ cytotoxic T lymphocyte response.

CD8+ cytotoxic T lymphocytes (CTLs) play an important role in containment of virus replication in primary human immunodeficiency virus (HIV) infection. HIV's ability to mutate to escape from CTL pressure is increasingly recognized; but comprehensive studies of escape from the CD8 T cell response in primary HIV infection are currently lacking. Here, we have fully characterized the primary CTL response to autologous virus Env, Gag, and Tat proteins in three patients, and investigated the extent, kinetics, and mechanisms of viral escape from epitope-specific components of the response. In all three individuals, we observed variation beginning within weeks of infection at epitope-containing sites in the viral quasispecies, which conferred escape by mechanisms including altered peptide presentation/recognition and altered antigen processing. The number of epitope-containing regions exhibiting evidence of early CTL escape ranged from 1 out of 21 in a subject who controlled viral replication effectively to 5 out of 7 in a subject who did not. Evaluation of the extent and kinetics of HIV-1 escape from >40 different epitope-specific CD8 T cell responses enabled analysis of factors determining escape and suggested that escape is restricted by costs to intrinsic viral fitness and by broad, codominant distribution of CTL-mediated pressure on viral replication.

Mapping sites of positive selection and amino acid diversification in the HIV genome: an alternative approach to vaccine design?

A safe and effective HIV-1 vaccine is urgently needed to control the worldwide AIDS epidemic. Traditional methods of vaccine development have been frustratingly slow, and it is becoming increasingly apparent that radical new approaches may be required. Computational and mathematical approaches, combined with evolutionary reasoning, may provide new insights for the design of an efficacious AIDS vaccine. Here, we used codon-based substitution models and maximum-likelihood (ML) methods to identify positively selected sites that are likely to be involved in the immune control of HIV-1. Analysis of subtypes B and C revealed widespread adaptive evolution. Positively selected amino acids were detected in all nine HIV-1 proteins, including Env. Of particular interest was the high level of positive selection within the C-terminal regions of the immediate-early regulatory proteins, Tat and Rev. Many of the amino acid replacements were associated with the emergence of novel (or alternative) myristylation and casein kinase II (CKII) phosphorylation sites. The impact of these changes on the conformation and antigenicity of Tat and Rev remains to be established. In rhesus macaques, a single CTL-associated amino substitution in Tat has been linked to escape from acute SIV infection. Understanding the relationship between host-driven positive selection and antigenic variation may lead to the development of novel vaccine strategies that preempt the escape process.

Intracellular delivery of antibodies using TAT fusion protein A.

Internalization of antibodies into mammalian cells is a useful method for analyzing and regulating cellular function. In this study, we developed a novel method for the delivery of antibodies into cells using the TAT-fused protein. This fusion protein consists of two functional domains, the protein transduction domain of HIV-1 TAT and the B domain of staphylococcal protein A (SpA), which has an ability to bind to the IgG. The TAT-SpA fusion protein was mixed with fluorescence-labeled rabbit IgG and added to cells. The internalization of antibody was analyzed using confocal microscopy and flow cytometry in living cells. As a result, fluorescence-labeled IgG with the TAT-SpA fusion protein was observed intracellularly. Flow cytometry results demonstrated time course and dose dependence relationships of antibody internalization. These results suggest that the TAT-SpA fusion protein can be a useful reagent for the delivery of antibody into cells.